The story of Kary Mullis and the invention of PCR

Some stories from science are fascinating. The story of Kary Mullis and his eureka moment which led to the invention of the polymerase chain reaction (PCR) is one such story which I love reading about.

For people not in the know, PCR is a path-breaking technique in molecular biology which, in the words of The New York Times, virtually divides biology into two epochs: before PCR and after PCR (1). PCR can enhance the detection of very minute quantities of DNA . The technique allows amplification of this DNA in order to study it further. The credit of this invention goes to Kary Banks Mullis.

Kary Banks Mullis grew up in a farm in a rural area, where he acquired his skills of observation. He says he designed rockets with his adolescent companions and loved tinkering in his high school labs. He entered graduate school at the University of California, Berkeley in 1966. He initially studied iron metabolism and transport in bacteria with specialized proteins (called siderophores) in Dr Joe Neilands’ biochemistry research laboratory.

But he was a very interesting character. He took several courses in music, anthropology, math, physics and sociology outside of the biochemistry department. While his thesis was complete, because he had not taken any courses in molecular biology, there were problems with the acceptance of his thesis (2). As the story goes, he decided to show his assessors that he knew enough about the scientific method, by publishing an astrophysics paper titled “The cosmological significance of time reversal” in the prestigious journal Nature! That wayward publication and achievement came to his rescue in ultimately earning his PhD in biochemistry in 1972.

But this man was a rolling stone. After obtaining his PhD, he left science to write fiction. Then he quit to join a medical school in Kansas City to become a biochemist. He left that job to manage a bakery for two years. He eventually returned to science and biotechnology, and to Berkeley, where he first studied rat brains, and later, began to study DNA synthesis.

He was hired by Cetus in 1979. It was there that he worked with oligonucleotides. Mullis went on to win a Nobel Prize in Chemistry in 1993 for his invention of the polymerase chain reaction. I found it fascinating to read his Nobel Lecture and his article in the Scientific American where he describes the moment when the revelation came to him on a moonlit drive through the mountains of California.

As the story goes, one Friday evening, Kary Mullis was driving in Mendocino County along the California Highway 128 from Berkeley to Anderson Valley to his cabin in the woods. His chemist girlfriend, Jennifer Barnett, was asleep on the adjacent seat. He was thinking:

Wouldn’t it be simple enough to put two oligonucleotides into the reaction instead of only one, such that one of them would bind to the upper strand and the other to the lower strand with their three prime ends adjacent to the opposing bases of the base pair in question. If one were made longer than the other then their single base extension products could be separated on a gel from each other and one could act as a control for the other. I was going to have to separate them on a gel anyway from the large excess of radioactive nucleosidetriphosphate. What I would hope to see is that one of them would pick up one radioactive nucleotide and the other would pick up its complement. Other combinations would indicate that something had gone wrong. It was not a perfect control, but it would not require a lot of effort.

He liked the idea of a control that was nearly free in terms of cost and effort. He continued to think about it and about things that could possibly go wrong.

What if the oligonucleotides in the original extension reaction had been extended so far they could now hybridize to unextended oligonucleotides of the opposite polarity in this second round. The sequence which they had been extended into would permit that. What would happen?

EUREKA!!!! The result would be exactly the same only the signal strength would be doubled. EUREKA again!!!! I could do it intentionally, adding my own deoxynucleoside triphosphates, which were quite soluble in water and legal in California. And again, EUREKA!!!! I could do it over and over again.

He stopped his car, found some paper and a pen, and made some calculations. He confirmed that if he had cycled this reaction thirty times he would be able to get the sequence of a sample with an immense signal and almost no background. As he drove further, his idea became more clear. As he says:

“I had solved the most annoying problems in DNA chemistry in a single lightning bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily.

Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it.”

Excited he tried to wake up Jennifer, but she wouldn’t wake up. Sometimes I feel Jennifer should be given credit for dozing off and letting him think uninterrupted!

He couldn’t sleep well over the weekend. On Monday, he searched his library and found nothing in literature about amplifying DNA by repeated reciprocal extension of two primers hybridized to separate strands of a particular DNA sequence.

Worse, none of his friends or colleagues were excited with his idea. Mullis had always had wild ideas, and they thought this must just be another one of them. But this was different. One or two of his technicians displayed polite interest. It was the time when his love life was crumbling. And when he and Jennifer fell out, both Mullis and his ideas suffered her scorn together.

Several sporadic experiments and three months later, Mullis could set up his first successful PCR reaction. Interestingly neither Nature nor Science accepted his Nobel Prize worthy manuscript and he was rather upset about it. He later got it published in Methods in Enzymology.

Unfortunately his personal life suffered the brunt of his laboratory work. Jennifer left him. He talks of personal grief overshadowing the excitement of success:

In Berkeley it drizzles in the winter. Avocados ripen at odd times and the tree in Fred’s front yard was wet and sagging from a load of fruit. I was sagging as I walked out to my little silver Honda Civic, which never failed to start. Neither Fred, empty Becks bottles, nor the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome.

***

Like all great people, Mullis had a streak of eccentricity in him. In 1992, he founded a company called ‘Star Gene’ intending to make and market jewelry which contained amplified DNA of famous celebrities, rockstars and athletes, like Marilyn Monroe and Elvis Presley! He has been described as an impatient and impulsive researcher who finds routine lab work boring. He wasn’t just eccentric- he could be outrageous. He refuses to believe that HIV is the cause for AIDS. In fact, minutes before his Nobel lecture, he told the organizer that he’d rather talk about why HIV wasn’t the causative organism of AIDS instead of speaking of PCR! In his autobiography, Dancing Naked in the Mind Field, Mullis writes about his forays with drugs and women, his belief in astrology and his encounter with an extra terrestrial fluorescent raccoon.

It takes all kinds of people, including those who think out of the box, to advance science. Kary Mullis is one of them.

(Featured image is from his 2009 TED Talk)